Fluidized bed detector for continuous, ultra-sensitive detection of biological and chemical materials

ABSTRACT

The present invention is generally directed to a fluidized bed detector for continuous detection of biological and chemical materials comprising a fluidized bed of detecting elements suspended in a continuous flow system wherein the detecting elements remain in the system when a first force trying to move the detecting elements to the bottom of the system is balanced with a second opposing force of a flowing gas or liquid trying to move detecting elements to the top of the system and wherein the presence of a target molecule in the flowing gas or liquid disrupts the balance of the first and second forces causing the detecting element to exit the system. The release of the detecting element indicates the presence of the target molecule and may be captured, concentrated, or both for further evaluation by other assays or other means. Also disclosed is the related method of detecting biological and chemical materials using a fluidized bed detector.

PRIORITY CLAIM

The present application is a divisional application of U.S. applicationSer. No. 12/618,180 filed by David A. Kidwell on Nov. 13, 2009 entitled“FLUIDIZED BED DETECTOR FOR CONTINUOUS, ULTRA-SENSITVE DETECTION OFBIOLOGICAL AND CHEMICAL MATERIALS,” which was a non-provisionalapplication that claimed the benefit of U.S. Provisional Application No.61/114,623 filed on Nov. 14, 2008 by David A. Kidwell, entitled“FLUIDIZED BED DETECTOR FOR CONTINUOUS, ULTRA-SENSITVE DETECTION OFBIOLOGICAL AND CHEMICAL MATERIALS,” the entire contents of each areincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to detectors based on bindingevents and, more specifically, to fluidized bed detectors forcontinuous, ultra-sensitive detection of biological and chemicalmaterials.

2. Description of the Prior Art

There is a continued need for sensors for materials such as chemicals,bacterial toxins, and viruses at small concentrations in a large volumeof media, where media may be defined as a solvent, such as water, asolid, or a gas or a mixture of media such as food which contains allthree states. Some substances that affect biological function are known,so that specific detectors are possible, and some are unknown, so onlyliving organisms can be the sentinel detector. Much effort has been andis being spent on assays for these materials that employ living cells,such as bacteria, mammalian cells, or small organisms as the detectionelement. The classic example is the use of canaries in mines to detectharmful gases. Because the canaries have a smaller body mass than humansand a higher respiratory rate, they tend to succumb first if harmfulgases are present. Live bioassays have a number of issues that limittheir use. (1) The living organism must be kept alive. This requiressome care and feeding even when the “sensor” is not being actively used.(2) Live bioassays that use complex organisms (such as canaries) mayrespond to other factors that do not cause appreciable concern forhumans, such as temperature and transportation stress. (3)Interpretation of the response of the complex organism may be difficultin varying environments. For example, why did the canary die? Was it atoxic substance in the air, heat stress, or an avian virus that onlyaffects canaries but not humans or other species? (4) For cell-basedlive assays, the cells also must be kept alive and more importantly sometransduction mechanism must be engineered into the cells to identify thethreat. For example, the transduction mechanism may be the production ofgreen fluorescent protein or firefly luciferase in response to certainsubstances. In these cases, the cells would be monitored for an increasein fluorescence or light emission upon addition of suitable substrates.Although such a cell-based, live bioassay can be useful, each organismmust be engineered to respond to a unique substance, which can be adaunting task if the agent in question does not have a known signalingpathway.

If the agent in question is known, specific detectors are possible. Theymay be either based on immunoassays, where the antibodies or otherspecific binding molecules such as peptides can act as the recognitionelement, or nucleic acid detectors where specific sequences of DNA orRNA are sought. The major issue with many specific detectors is that thevolume of liquid or air testable is quite small so that apreconcentration step is required. Without this preconcentration step,the likelihood of detection of a target species decreases with theconcentration and the volume of material needed for the test (see FIG.1). Because of this preconcentration step, the testing technology isoften a grab-and-sample type of test, i.e., the preconcentrator is runfor a given amount of time, a sample of the concentrate is taken, andthe sample tested. This process can be repeated on a periodic time scalebut as most tests are single-use tests where consumption of reagents canbe substantial so that the frequency of testing is often limited. Mostimmunoassays are single use; an example being the lateral-flowimmunoassays used in home-pregnancy testing. An exception isdisplacement assays such as the flow-immunoassay described in Kusterbecket al., “A continuous-flow immunoassay for rapid and sensitive detectionof small molecules,” Journal Of Immunological Methods, 135, 191-197(1990), the entire contents of which are incorporated herein byreference. Displacement assays, although proposed here as well, are notas sensitive as completion or sandwich immunoassays due to kinetics andantibody affinity. For high sensitivity, the antibody binding constantmust be as high as possible. However, tight binding means slow releaseso that the displacement with a test antigen either is slow or requireshigh levels of antigen.

U.S. Patent Application 20040009529 by Weimer et al. (Jan. 15, 2004),the entire contents of which are hereby incorporated by reference,describes a process for the capture of antigens on beads in aflow-though module. This is not a continuous assay as the capturedantigens must be detected, (for example with an enzyme-linked antibody)in a grab and sample mode. The particles are not released upon bindingto the antigen. This system is similar to the BEADS (BiodetectionEnabling Analyte Delivery System) sample preparation technologydescribed in the next paragraph.

U.S. Pat. No. 6,506,584 to Chandler et al. (Jan. 14, 2003), U.S. Pat.No. 6,159,378 to Holman et al. (Dec. 12, 2000), and U.S. Pat. No.6,136,197 to Egorov et al. (Oct. 24, 2000), the entire contents of eachare hereby incorporated by reference, describe PNNL's BEADS(Biodetection Enabling Analyte Delivery System) sample preparationtechnology. Although claimed as a fluidized bed for rapid kinetics, PNNLdoes not use a force field to retain the particles but rather a complexcapture and release system for the particles to maintain them in afluidized state. Like the Weimer application, the BEADS system is more agrab and sample system. Furthermore, in the way that the particles areretained (like a simple screen), the BEADS system cannot handle complexmedia, such as food, as it will quickly plug.

U.S. Pat. No. 6,153, 113 to Goodrich et al. (Nov. 28, 2000), the entirecontents of which are hereby incorporated by reference, uses acontinuous centrifuge and binding particles. However, it is not adetector and it does not release the particles upon binding. Rather theparticles only act as a capture medium to remove a selective bloodcomponent from the incoming fluid with purification of the fluid (bloodin this case) as the goal.

U.S. Pat. No. 4,939,087 to Van Wie et al. (Jul. 3, 1990), the entirecontents of which are hereby incorporated by reference, describes theuse of a centrifugal reactor for culturing cells and harvestinghigh-valued proteins in a continuous manner. This patent shows that acentrifugal reactor can maintain living cells on a long-term basis.However, it is not used as a detector.

BRIEF SUMMARY OF THE INVENTION

The aforementioned problems are overcome in the present invention whichprovides a fluidized bed detector for continuous detection of biologicaland chemical materials comprising a fluidized bed of detecting elementssuspended in a continuous flow system wherein the detecting elementsremain in the system when a first force trying to move the detectingelements to the bottom of the system is balanced with a second opposingforce of a flowing gas or liquid trying to move detecting elements tothe top of the system and wherein the presence of a target molecule inthe flowing gas or liquid disrupts the balance of the first and secondforces causing the detecting element to exit the system. The release ofthe detecting element indicates the presence of the target molecule andmay be captured, concentrated, or both for further evaluation by otherassays or other means. Also disclosed is the related method of detectingbiological and chemical materials using a fluidized bed detector.

The present invention generally relates to a continuous flow sensorbased on a fluidized bed. This sensor can operate with living cells orinert particles as the detecting elements for target materials. In manymodes of operation, no routine maintenance would be necessary assupplies are not depleted unless a detection event occurs. The sensorcan operate with high flow rates, which mitigate the need for separatelarge, power-hungry pre-concentration schemes. Such a sensor should findwide deployment in the monitoring of the nation's air, water, food, andagricultural areas for the presence of biological and chemical threats.

Some of the advantages of the Fluidized Bed Detector are:

-   -   It can detect low target numbers—a single binding event/mL is        possible because single-particle labels are readily detectable.    -   It can be multiplexed by having different labels on each        particle corresponding to different antibodies/DNA.    -   It can handle high flow rates (liters/hour), which reduces the        need for pre-concentrators (some fluid can be re-circulated, if        necessary).    -   It is a continuous flow system—no wait for the detection        event—detection in minutes.    -   It requires no addition of reagents—once started the particles        are retained—some fluid make-up (i.e. saline) may be necessary        in certain modes of operation.    -   The rapid particle collisions allow shear (the magnitude of        which can be controlled) thereby providing selectivity over        non-specific agglutination.    -   It can be operated in the presence of a high particle count        media as the flow channels are not restricted nor are filters        necessary to remove debris, which avoid plugging or replacement        of the filters.

The fluidized bed detector (FBD) of the present invention is quitedistinct from xMAP® technology pioneered by Luminex using a Coultercounter in that xMAP is not continuous and cannot handle very largenumbers of particles nor a large volume of solution and must count manyparticles rather than the small number in the FBD. Additionally, thexMAP technology does not concentrate species, once detected. In oneversion of the xMAP system, the particles are fluorescently labeled andcoated with fluorescent dyes such that the classes of particles aredistinguishable. For example, two fluorescent dyes may be employed thathave distinct emission characteristics. The ratio between the two dyesallows different classes of particles to be distinguished. The classesof particles have antibodies on the surface. The test antigen may befluorescently labeled. After mixing with the test antigen, the wholemixture is passed though a Coulter counter, where each particle isexamined. The two labels tell the class of particle and the remainingfluorophor would be present if the antigen were present. Note in thisscheme, a large number of particles need to be counted as only a fewwould have the antigen. Additionally, the antigen must be labeled withanother fluorophore, which could be a second antibody, and the reagentsare used for each assay and cannot be easily recycled. Furthermore, theprinciple of the Coulter counter is to pass the particles through arelatively small hole to separate them one at a time so that each may beinterrogated. Such a hole is easily plugged by particles in the testmatrix and the therefore, solids or slurries cannot be handled.

The FBD could use a similar particle-labeling scheme to Luminex formultiplexing, but that is where the similarity ends. No small holes arenecessary to sort the particles as the release per unit time should belimited. Also, the FBD may use two distinct fluorophore containingparticles that cross-link. In that case, only particles would be countedthat have both fluorophores present in the complex. This scheme willprovide better specificity as it avoids counting particles that leakfrom the system as leaking particles would have only one distinctfluorophore pattern present.

These and other features and advantages of the invention, as well as theinvention itself, will become better understood by reference to thefollowing detailed description, appended claims, and accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the probability of detection a bacterial spore versessample volume. The lines enclose a probability of from 1% to near 100%detection window. For example, if the sampling volume was 1 μL, to reach1 species in that volume one would need to have over le6 species/L (seeK. E. Petersen et al., “Toward Next Generation Clinical DiagnosticInstruments: Scaling and New Processing Paradigms,” BiomedicalMicrodevices, 1, 71-79, (1991) the entire contents of which areincorporated herein by reference).

FIG. 2 shows two examples of particles interacting with biomolecules:FIG. 2( a) shows a particle capturing a bacteria wherein the complex islost from the top as density decreases and FIG. 2( b) shows DNA linkingtwo particles wherein the complex is lost from the bottom as densityincreases.

FIG. 3 is a schematic diagram of the fluidized bed detector, which hasno rotating seals. The hoses are protected from twisting by a counterrotation at the base. (See, e.g., U.S. Pat. No. 6,153,113 to Goodrich etal. (Nov. 28, 2000), U.S. Pat. No. 4,425,112 to Ito (Jan. 10, 1984), andU.S. Pat. No. 4,114,802 to Brown (Sep. 19, 1978), the entire contents ofeach are incorporated herein by reference.)

FIG. 4 shows (a) one embodiment of the fluidized bed detector; (b) anembodiment that could be a commercial system, which is the inside of theAmicus Separator (one example is the Amicus Separator at:

http://www.baxterfenwal.com/jsp/products/productDetail.jsp?prodid=159&familyid=36);and (c) a side view of the commercial system showing the precisiongearing used for faster speeds and balance.

FIG. 5 shows a test done with iron-oxide particles. Iron-oxide particlesas model “dirt” flowed through the cell while red-dyed polystyrene beadswere retained. Although more dense, the “dirt” being too small (1-2 μm)was not retained under the conditions used. FIG. 5( a) shows the browndirt without dyed polystyrene particles. FIG. 5( b) shows polystyreneparticles with the “dirt” flowing through as a brown haze in the top ofthe flow-cell. FIG. 5( c) shows the same polystyrene particles when thedirt was removed. The pictures were taken with a strobe light as thecentrifuge was continually moving, and the flow was also continuous tofluidize the particles.

FIG. 6 shows some representative flow cell designs. Flow cells werefilled with a colored dye to highlight the shape. FIG. 6( a) shows atwo-dimensional flow cell, partially filled with a liquid to highlightthe cylindrical shape. FIGS. 6( b)-(j) have square sides.

DETAILED DESCRIPTION OF THE INVENTION

The Fluidized Bed Detector (FBD) of the present invention is basically asystem containing detecting elements wherein the detecting elements aresuspended in the system using electrical fields, magnetic fields,acceleration forces, or any combination thereof to retain the particlesagainst a counter-flow of a fluid such as a liquid or gas containing thetarget of interest. In one embodiment, the system could be a centrifuge(to increase sedimentation rates) using centrifugal force tocounterbalance the force of the fluid flow. Detection particles areinitially introduced into the analysis chamber by flowing them into thebottom while the chamber is spinning. The forces acting in the FBD canbe mathematically modeled with equations 1-3. The particles are retainedin the spinning chamber by the balancing of two forces: the centrifugalforce (equation 1) (this could also or alternatively be a magnetic orelectrical field or a gravitational force), which causes the particlesto exit the outside (bottom) of the spinning chamber, and the fluid flow(equation 2), which causes the particles to exit the inside (top) of thechamber. When these two forces are in balance (equation 3), no particlesexit the chamber—only the flowing liquid (which may contain the targetsof interest) exits the top and bottom. When there is a target moleculein the fluid flow, the balance of the two forces is disrupted causingthe detecting element to exit the chamber. The balance of the forces canbe disrupted by a cell being killed (the cell is the detecting element),by the binding of the target to the detecting element, the cross linkingof two particles, or two particles previously cross-linked breakingapart.

These two forces have different physical sources. Movement by thecentrifugal force depends on the density of the particles relative tothe fluid. Movement by the fluid flow depends on the average face area(surface area projected along the fluid flow) of the particles. Duringthe detection event, the particles change their density relative totheir face area. (Face area is the area projected into the flow. For asphere this area is just a circle with the same diameter as the sphere.For a cylinder, it is a complex function of the tumbling rate and endarea.) Two examples of how interacting particles can change theirdensity relative to face area are shown in FIG. 2. The cause of thischange depends on the type of assay and particles being employed. Oncethis change occurs, the centrifugal and flow forces are no longerbalanced and the particle leaves the centrifugal chamber (either thoughthe bottom or top) where it is detected by some means, for exampleabsorption, fluorescence, change in magnetic signature (such as amagnetic particle changing the impedance of a coil), colorimetric assay,etc. Because single particles can be readily counted and measured, achange in a single particle, of the many suspended in the chamber, maybe detectable. Unlike many assays that rely on binding of antibodies ornucleic acids to surfaces and binding of the target to those species,the fluidized bed is well mixed by the incoming flowing stream so thatkinetics are rapid. Additionally, a large excess of particles may bepresent allowing more rapid kinetics due to concentration effectswithout compromising sensitivity. (For example, in competitiveimmunoassays, the greatest sensitivity is found when the concentrationof the antibody is one-half the concentration of the analyte (due toantibodies having two binding sites). As immunoassay kinetics requirestwo entities to interact, the reaction rate is dependent on both theconcentration of the analyte and antibody (a second order reaction).Therefore, the time for interaction must increase as the inverse squareof the analyte concentration.)

$\begin{matrix}{{{Force}\mspace{14mu} {on}\mspace{14mu} {particle}\mspace{14mu} {due}\mspace{14mu} {to}\mspace{14mu} {centrifugal}\mspace{14mu} {force}\text{:}}{F_{centrifugal} = {{{mr}\; \omega^{2}} = {\frac{{TTd}_{particle}^{2}}{6}\left( {\rho_{particle} - \rho_{medium}} \right)\left( {r\; \omega^{2}} \right)}}}} & (1) \\{{{Force}\mspace{14mu} {on}\mspace{14mu} {particle}\mspace{14mu} {due}\mspace{14mu} {to}\mspace{14mu} {fluid}\mspace{14mu} {flow}\text{:}}{F_{StokesDrag} = {3\; {TT}\; \mu \; d_{{particle}^{v}{particle}}}}} & (2) \\{{{Balanced}\mspace{14mu} {when}\mspace{14mu} {equal}\text{:}}{v_{particle} = {\frac{d_{particle}^{2}}{18}\left( {\rho_{particle} - \rho_{medium}} \right)r\; \omega^{2}}}{d_{particle} = {diameter}}{\rho = {density}}{\omega = {{radians}/\sec}}{v = {velocity}}{\mu = {viscosity}}{r = {{centrifuge}\mspace{14mu} {radius}}}} & (3)\end{matrix}$

FIG. 3 shows the basic concept for the FBD using labeled particles. Theparticles may be either living cells or inert particles. FIG. 4 showsthe model FBD constructed for preliminary testing and the inside of acommercial unit used for blood processing. The commercial unit employsbalances and precision gears to rotate the upper stage at twice therotational speed as the arm.

A preliminary system used belts and a variable-speed drill motor to turnthe main centrifuge. The speed was controlled with a laboratory Variacand not automatically stabilized (the user needed to make smalladjustments until the desired speed was obtained). The speed wasmonitored using a magnetic pick-up Reed switch with a permanent barmagnet mounted on the rotor arm. The signal from the switch triggered astrobe light, which allowed movies to be made of the flow, and was alsofed into a RS232 port of a computer. The signal into the RS232 portprovided the start bit for pseudo-character (basically read as the ASCIINull character), which was read by the computer. The timing betweencharacters was measured and averaged every few seconds (the programallowed variable averaging) to report the RPMs of the centrifuge. Withthis preliminary system, 1000 RPM movement could be generated. With thecenter of the cell at an average distance of 19 cm, this would produce112 g force on the particles at 1000 RPM. Better balancing of thispreliminary design may allow faster speeds and is important as the gforce increases as the square of the rotational velocity. The higher theg force, the better the resolution between two objects. Commercialsystems can achieve over 6000 RPM.

One advantage of the FBD system of the present invention over otherfluidized bed collection schemes is that debris does not have to beseparated before the sample is tested. Many test samples, such as food,contain particles or debris that are not of interest. For mostflow-though assays, these particles must be separated either withfilters or by centrifugation before the sample is assayed or theparticles will interfere. The FBD does not have this requirement. Thereare no filters, small paths, or sharp angles to plug in the FBD. Thepath is continuous. Only those particles meeting the density-size-flowbalancing will be retained. By using particles as the detection elementin the FBD they have the advantage over living cells in that they can beengineered to have a wide range of densities that can then handle a widerange of fluid flows and discriminate against nuisance particles. Forexample, FIG. 5 shows the flow of colloidal ion particles (used asmodels of dirt and for their color) through the FBD while retaining thelatex beads. Milk (high protein content and homogenized particles),diluted tomato paste, and diluted ketchup were run though the FBD whileretaining the latex beads being tested as sensors. The tomato paste andketchup left some strands of pulp indicating that a higher flow withdenser sensor particles would have been advantageous. The FBD may bevery useful in food testing for bacteria as a large number of samplescan be tested quickly in a flow system and allowing isolation ofparticles that may be cultured for confirmation of the presence of acertain bacterial species.

Another advantage of the FBD, is that upon release of the detectingelement, the released detecting element selectively can be captured,separated, concentrated, analyzed, or any combination thereof. When thedetecting element is released, it carries with it the target material.When detected, the detecting element can be shunted selectively into acollection system for further analysis or disposal where the othercomponents of the test matrix are shunted for disposal or furtheranalysis. This selective separation ability provides the opportunity toconcentrate targets from large volumes as part of the initial warningsystem.

Particle Based System

The FBD system has flexible requirements for the labels used in thedetector. One class of materials could be inert materials such as eitherpolymer or glass based beads. Having the materials homogeneous indiameter and density makes construction easier. The beads haveantibodies, nucleic acids, complexes, or any combination thereof ontheir surfaces, which in the presence of a target molecule eithercross-links two or more particles (sandwich assay) or breaks a complexapart (displacement assay). The term antibodies can refer to a number ofprotein binding molecules such as antibodies, antibody fragments,enzymes, or engineered peptides that selectively recognize othermolecules. The term nucleic acids is being used to encompass a widerange of DNA or RNA selective binding molecules—they may also be DNA orRNA bases with non-conventional backbones such as peptide nucleic acids;however, DNA or non-conventional backbones are preferred over RNA as itis more stable in solution. The term complexes can refer to moleculesthat recognize other small species such as metal ions. Examples may beEDTA, which is selective for calcium or six histidines, which isselective for nickel. For these complexes, the binding of the metal ionis unlikely to change the particle density sufficiently to be useful.Instead, the target metal ion will displace a ligand attached to alarger molecule or particle in a displacement type assay.

Cross linking of two particles changes the average face area to densityratio and the complex will flow out the bottom of the spinning chamberwhere it is detected by some means, for example fluorescence. Thus, thepresence of a large target molecule/species (virus, bacterium, or DNA)that can form a sandwich assay will be detectable by the release oflabeled particles. Note that the target is not labeled, so raw materialcan be analyzed without preparatory steps. The release of labels(fluorescent latex spheres in one configuration) indicates the presenceof a given target. Small molecules also can be detected by disrupting(displacement assay) a preformed complex that has the correct buoyancywhen two particles are bound together but not when separated. Thelabeled particles of the disrupted complex would flow out the top (hencea detector on that outlet).

Unlike normal agglutination assays, a single binding event can bedetectable. Additionally, unlike surface assays, the fluidized bed iswell mixed by the incoming flowing stream so that kinetics ofinteraction is rapid. Because the measurements are made outside thechamber, a large excess of particles may be present on the inside asthese are never seen by the detector, which may be a Coultercounter-like system.

Cell-Based System Live System

For sentinel systems, it is often useful to have living organismspresent as test subjects. Bacteria or human cells are not ideal becausethey may be killed or react to any number of materials that are notacutely toxic, such as high salt concentrations or pH changes. However,cells are much easier to keep alive than higher order organisms and morecan be fit in a given space. Consider the inert particles, discussedabove, as replaced by cells. The basic concept is to continuouslymaintain cells or bacteria in the FBD while outside nutrients and testcompounds are introduced. Fluidized beds have been considered for justsuch a scheme as they allow continual harvesting of valuable proteinsthat may be secreted by the cells and a constant monitoring of the media(see U.S. Pat. No. 4,939,087 to Van Wie et al., Jul. 3, 1990, the entirecontents of which are incorporated herein by reference). The fluidizedbed allows greater cell densities to be achieved and faster growth.Although much more complicated than inert particles, living cells couldbe used in several ways:

Release of Materials

The cells are maintained by their density and size in the system andrespond by changing their protein coat or releasing materials whenoutside compounds trigger some biochemical process. Almost any type ofcell response that is selective in the changing environment and occurson the reporter-cell surface can be detected by this system. Forexample, when cells die, there density decreases and they would flow outof the FBD. Thus, even responses to viruses would be detectable. Thereleased materials would be detected in the flowing stream by additionof antibodies or by engineering the released materials to be inherentlyfluorescent or by adding a dye to the exit stream that selectivelylabels the target cells.

Instead of detecting the released materials in the stream one couldcombine the living cells with inert particles. If the reporter cellsexcreted a protein or other large molecule into the medium, this couldbe detectable by crossing-linking the reporting labels. For example, ifthe labels were particles of a similar density to the cells andcontained antibodies to an excreted protein, say anti-luceferase, thenthe excreting of the luceferase by the cells would cross-link thereporter particles and cause them to be released (In this case, thereporter particles would likely be latex beads, which are predominatelyspherical. These complexes are released from the centrifugal reactorbecause the centrifugal force is no longer counterbalanced by theincoming flowing liquid. The force that the flowing liquid force exertsis based on the face area exposed to the flow where as the centrifugalforce acts on the density (which is different, generally higher, thanthe incoming media otherwise the particles would not move). Cross-linkedparticles have a higher density to face surface area than do singleparticles because they do not always face parallel to the incomingliquid (i.e. one particle shields the other). Thus, they will move tothe bottom of the FBD.

As envisioned with the FBD, even the presence of DNA or other largemolecules that do not affect the cell population could be detectable. Inthis case, there would be no biological amplification and only areliance on the cross-linking of the particles would occur.

Release of Dead Cells

Cells that die tend to have a different density then living cells andwould be swept from the FBD. The living cells could be stained with adye upon release and the fluorescence of the stain monitored toessentially count the release vs. time. If a major increase in releaseis noted, then the death of the cells in the FBD must be from somecause—toxin or virus that would need to be investigated further. Onecould distinguish the release of cells from the FBD vs. cells present inthe feed water by staining or more specifically by antibodyinteractions. The antibody interactions would allow identification of anumber of released cells as the antibodies could be specific to acertain cell type. For example, the cells could be stained with alive-dead stain such as the SYTOX Green Stain sold by Molecular Probes.This stain does not stain cells that have intact membranes. Theantibodies may be labeled with a fluorescent dye such as Rhodamine. Onlythose cells that had both fluorophores present would be consideredcounted and released. Unfortunately, continual addition of antibodies isexpensive unless the antibodies were recovered in the flowing fluid andmay not be necessary if the incoming fluid has few cells present thatwill stain. Stains are relatively cheap. To save resources, one couldprestain the incoming fluid but that gets more complex as the stainwould be present in the FBD chamber. To be successful, a prestain couldbe designed to change some property that increases the staining and thenreduces it. For example, the incoming test fluid may be adjusted to alow pH, which could increase staining. Then the system is buffered toneutrality when it flows though the FBD chamber. Another stain is addedon the outlet that stains everything at neutrality. Only those cellswith the second stain and without the prestain would be considered fordetection. Alternatively, the incoming fluid may be passed through abed, such as activated charcoal, to remove the stain but not thepotential target toxins. One could also engineer the cells to produce afluorescent protein and retain it to identify the cell. Each cell typewould be engineered to produce a different fluorescent protein.

Release of Cross-Linked Cells

Some cellular systems have on their surface receptors and can cross-linkin the presence of a given antigen. These receptors may be engineered,as in phage display protein libraries, to be similar to antibodies intheir binding, recognition, and specificity. Cross linking of two cellscauses their release from the FBD chamber. This system is preferable forease of detection, but is really no better than the use of inertparticles coated with antibodies, as discussed above. In fact, one coulduse killed cells as the inert particles for cost considerations and justprestain them or have them engineered to be fluorescent.

Flow Cell Design

A number of different flow cells were tested to achieve Laminar flow.Examples are shown in FIG. 6. If the cells had an inlet that wentparallel to the axis, i.e. directly into the bottom, the flow was veryerratic. Having the inlet with a curved flow helped. Also, having aninlet with a pressure chamber (as shown for the long cell in FIG. 6 g)helped but the chamber tended to entrap the particles used for labels.The inlet on the cell in FIG. 6 j was the optimum found for a uniformflow. The centrifugal force decreases as the radius decreases whereasthe force due to fluid flow is constant. The flow cell should be taperedto provide stability in retention of the particles as the taperdecreases the fluid flow in proportion to the radius. A slightly greaterincrease in taper than predicted on a quadric exponential was optimum.This shape is shown in FIG. 6 e. Additional cells with both a taper anda bulb, for particle storage, allow different shear forces depending onthe position in the cell.

The above descriptions are those of the preferred embodiments of theinvention. Various modifications and variations are possible in light ofthe above teachings without departing from the spirit and broaderaspects of the invention. It is therefore to be understood that theclaimed invention may be practiced otherwise than as specificallydescribed. Any references to claim elements in the singular, forexample, using the articles “a,” “an,” “the,” or “said,” is not to beconstrued as limiting the element to the singular.

What is claimed as new and desired to be protected by Letters Patent ofthe United States is:
 1. A fluidized bed detector for continuousdetection of biological and chemical materials, comprising a fluidizedbed of detecting elements suspended in a continuous flow system, whereinthe detecting elements remain in the system when a first force trying tomove the detecting elements to the bottom of the system is balanced witha second opposing force of a flowing gas or liquid trying to movedetecting elements to the top of the system, wherein the presence of atarget molecule in the flowing gas or liquid disrupts the balance of thefirst and second forces causing the detecting element to exit the systemwherein the release of the detecting element indicates the presence ofthe target molecule.
 2. The fluidized bed detector of claim 1, whereinthe detecting element is a living cell and the balance of the first andsecond forces is disrupted by the killing of the cell.
 3. The fluidizedbed detector of claim 1, wherein the balance of the first and secondforces is disrupted by the binding together of the target molecule withthe detecting element and thereby changing the density or face area ofthe resulting complex.
 4. The fluidized bed detector of claim 1, whereinthe balance of the first and second forces is disrupted by thecross-linking of detecting elements or the breaking apart of previouslycross-linked detecting elements.
 5. The fluidized bed detector of claim1, wherein the first force is magnetic, electrical, acceleration, or anycombination thereof.
 6. The fluidized bed detector of claim 1, whereinthe system is a continuous centrifuge and the first force is acentrifugal force.
 7. The fluidized bed detector of claim 1, wherein thedetecting elements are inert particles or living cells.
 8. The fluidizedbed detector of claim 1, wherein the detecting elements are polymerbeads, glass beads, or a combination thereof, wherein the surface of thebeads have antibodies, nucleic acids, complexes, or any combinationthereof.
 9. The fluidized bed detector of claim 1, wherein the detectingelements are cells that release materials when a target materialtriggers a biochemical process.
 10. The fluidized bed detector of claim1, wherein it is unnecessary to separate particles not of interest fromthe flowing fluid.
 11. The fluidized bed detector of claim 1, whereinthe release of the detecting element is detected by some means.
 12. Thefluidized bed detector of claim 11, wherein the detecting element isdetected by absorption, fluorescence, colorimetric assay, change inmagnetic signature, or any combination thereof.
 13. The fluidized beddetector of claim 1, wherein the released detecting element may becaptured, concentrated, or both for further evaluation.
 14. A method fordetecting biological and chemical materials, comprising: suspending afluidized bed of detecting elements in a continuous flow system,maintaining the detecting elements in the system by balancing a firstforce trying to move the detecting elements to the bottom of the systemwith an opposing second force of a flowing gas or liquid trying to movedetecting elements to the top of the system; and detecting the presenceof a target molecule in the flowing gas or liquid wherein the presenceof the target molecule disrupts the balance of the first and secondforces causing the detecting element to exit the system wherein therelease of the detecting element indicates the presence of the targetmolecule; wherein the detecting element is a living cell and the balanceof the first and second forces is disrupted by the killing of the cell.15. The method of claim 14, wherein the detecting elements are cellsthat release materials when a target material triggers a biochemicalprocess.
 16. A method for detecting biological and chemical materials,comprising: suspending a fluidized bed of detecting elements in acontinuous flow system, maintaining the detecting elements in the systemby balancing a first force trying to move the detecting elements to thebottom of the system with an opposing second force of a flowing gas orliquid trying to move detecting elements to the top of the system; anddetecting the presence of a target molecule in the flowing gas or liquidwherein the presence of the target molecule disrupts the balance of thefirst and second forces causing the detecting element to exit the systemwherein the release of the detecting element indicates the presence ofthe target molecule; wherein the system is a centrifuge and the firstforce is a centrifugal force.